SLC19A3 Antibody, Biotin conjugated

What is SLC19A3 and what is its biological function?

SLC19A3 (Solute Carrier Family 19 Member 3) encodes the Thiamine Transporter 2 (ThTr2), a protein that mediates high-affinity thiamine uptake, likely via a proton anti-port mechanism. The SLC19A3 protein plays a critical role in thiamine (vitamin B1) transport across cellular membranes, and research has confirmed it has no folate transport activity . Recent studies have expanded our understanding of its function, demonstrating that SLC19A3 also mediates H+-dependent pyridoxine transport . The protein has a calculated molecular weight of 56 kDa but is typically observed at 63-70 kDa in experimental applications, suggesting post-translational modifications . SLC19A3 function is essential for normal neurological development, as mutations in this gene cause biotin-thiamine responsive basal ganglia disease, a severe but potentially treatable neurological disorder .

How does biotin relate to SLC19A3 expression and function?

While biotin is not a known substrate for the SLC19A3 transporter, research has established a significant relationship between biotin status and SLC19A3 expression. Studies show that marginal biotin deficiency reduces SLC19A3 expression dramatically - to approximately 33% of baseline levels after 28 days of experimental biotin deficiency (p < 0.02) . This sensitivity to biotin status makes SLC19A3 expression a relatively sensitive indicator of marginal biotin deficiency compared to other biotin-related genes like MCCA, PCCA, PC, ACCA, ACCB, HCS, biotinidase, and SMVT, which showed less pronounced decreases to approximately 80% of baseline under the same conditions .

The mechanism behind this relationship reveals the intricate regulatory network that connects different vitamins: biotin supplementation leads to increased SLC19A3 expression, which may partially explain the clinical response observed in patients with SLC19A3 mutations when treated with biotin . This cross-talk between biotin and thiamine pathways highlights the complexity of vitamin transport and utilization systems in human physiology.

Biotin conjugation to SLC19A3 antibodies represents a strategic modification that enhances detection capabilities through the avidin-biotin complex (ABC) system. This conjugation offers several methodological advantages for researchers:

The biotin-conjugated SLC19A3 antibodies can bind with high affinity to streptavidin or avidin detection systems, providing signal amplification that increases sensitivity in applications like immunohistochemistry, ELISA, and flow cytometry . This is particularly valuable when detecting SLC19A3, which may be expressed at relatively low levels in certain tissues or under specific conditions, such as biotin deficiency states .

Researchers should note that when using biotin-conjugated antibodies to study SLC19A3 in relation to biotin metabolism or deficiency states, appropriate experimental controls must be implemented to account for potential interference from endogenous biotin, especially in biotin-rich tissues .

How can researchers optimize experimental conditions for SLC19A3 detection in various tissue types?

Optimizing SLC19A3 detection requires tissue-specific considerations due to variable expression levels and potential interference factors. For neurological tissue samples, where SLC19A3 plays a crucial role in thiamine transport and deficiency can lead to basal ganglia disease, researchers should consider the following methodological approaches:

RNA expression analysis can be effectively performed using primers targeting specific regions of the SLC19A3 transcript. Validated primer sets include:

• Forward: 5′-AGAGCAGAAACCCACATCAGAAAT-3′ (position 730)

• Reverse: 5′-GCTTGGTTTCAGGCTGTTCAG-3′ (position 808)

For protein detection in neurological samples, fixation with 4% formaldehyde followed by 0.2% Triton X-100 permeabilization has been validated for immunofluorescence applications, particularly in neuroblastoma cell lines like SH-SY5Y . When working with liver tissue samples, which show consistent SLC19A3 expression, researchers should utilize HepG2 cells as positive controls for antibody validation .

In experimental designs involving biotin deficiency models, it's critical to monitor expression changes over time, as SLC19A3 expression decreases to approximately 33% of baseline after 28 days of biotin restriction, making it a sensitive biomarker for marginal biotin deficiency states .

What methodological approaches can resolve conflicting data in SLC19A3 studies?

When facing contradictory results in SLC19A3 research, implementing a multi-modal verification strategy can help resolve discrepancies. Conflicting data may arise from species differences in SLC19A3 function, as evidenced by differential inhibitor sensitivity between human and murine THTR2 transporters. For example, metformin and fedratinib are at least nine times more potent inhibitors of human THTR2 compared to mouse THTR2 .

To address these species-specific variations, researchers should:

• Confirm antibody cross-reactivity across species of interest before initiating comparative studies. While some antibodies show reactivity with human, mouse, and rat samples , others may be species-restricted .

• Employ genetic validation through knockout models and rescue experiments. The transgenic model (SLC19A3int; TG) with human SLC19A3 inserted into murine Slc19a3 knockout provides a valuable tool for dissecting species-specific differences in transporter function .

• Implement complementary methodologies to validate protein expression patterns. When RNA-seq data suggests allelic silencing, whole-genome sequencing can reveal underlying genetic defects such as promoter deletions that might not be detected by standard sequencing approaches .

• For discrepancies in response to thiamine/biotin supplementation, researchers should consider allelic variations in SLC19A3. Novel mutations like the 45,049 bp deletion in the 5'-UTR region abolishing the promoter can significantly impact transporter expression and clinical response .

How can SLC19A3 antibodies help investigate the relationship between thiamine transport and biotin metabolism?

SLC19A3 antibodies provide valuable tools for elucidating the unexpected cross-regulation between biotin status and thiamine transport. To investigate this relationship, researchers can implement the following experimental approaches:

Dual-labeling immunofluorescence studies using biotin-conjugated SLC19A3 antibodies alongside markers for biotin-dependent carboxylases can visualize co-localization patterns in cells under varying biotin conditions . Time-course experiments monitoring SLC19A3 protein levels in response to controlled biotin deficiency and supplementation can reveal the kinetics of this regulatory relationship. Validated antibodies used at dilutions of 1:500-1:2000 for Western blot applications can quantitatively assess these changes .

Co-immunoprecipitation experiments using SLC19A3 antibodies can identify potential protein-protein interactions between SLC19A3 and biotin-dependent enzymes or transporters. For these applications, 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate is recommended for optimal results .

Chromatin immunoprecipitation (ChIP) experiments using antibodies against transcription factors known to regulate biotin-responsive genes can determine if SLC19A3 expression is directly regulated at the transcriptional level by biotin-responsive elements, explaining the observed 67% reduction in SLC19A3 expression during biotin deficiency .

How can researchers design experiments to study the differential expression of SLC19A3 compared to other biotin-related genes?

Designing robust experiments to compare SLC19A3 expression with other biotin-related genes requires careful consideration of sensitivity differences. Research has shown that SLC19A3 expression decreases to 33% of baseline during biotin deficiency, while expression of biotin-dependent carboxylases (MCCA, PCCA, PC, ACCA, ACCB) and related proteins (HCS, biotinidase, SMVT) decreases to only about 80% of baseline under the same conditions .

To accurately capture these differential responses, researchers should:

• Implement precise qRT-PCR assays with validated primer sets for each target gene. For example:

  • SLC19A3: Forward 5′-AGAGCAGAAACCCACATCAGAAAT-3′, Reverse 5′-GCTTGGTTTCAGGCTGTTCAG-3′

  • SMVT: Forward 5′-CACATCTTCATCCCCGTTTTCT-3′, Reverse 5′-TGAATCGAAGCTCCAGGTACTCA-3′

• Utilize 18s rRNA as an endogenous control to account for variability in initial RNA amounts and reverse transcription efficiency .

• Design time-course experiments that capture both early and late responses to biotin deficiency, as differential sensitivity may manifest at different time points.

• Consider the structural differences between carboxylase subunits. Research suggests that expression of biotin-binding chains of biotin-dependent carboxylases (like MCCA and PCCA) may be more responsive to biotin status changes than non-biotin-binding chains (MCCB and PCCB), which showed no significant change in deficiency states .

What methodological challenges arise when using biotin-conjugated antibodies in tissues with endogenous biotin and how can they be addressed?

Using biotin-conjugated SLC19A3 antibodies in biotin-rich tissues presents specific methodological challenges that must be addressed to ensure valid results. Endogenous biotin can compete with biotinylated antibodies for avidin/streptavidin binding sites, potentially creating false-negative results or reducing signal intensity .

To overcome these challenges, researchers should implement:

• Biotin blocking steps: Pre-treatment of samples with avidin followed by biotin blocking solution can mask endogenous biotin before applying the biotin-conjugated SLC19A3 antibody. This approach is particularly important when working with biotin-rich tissues like liver, kidney, and brain.

• Alternative detection systems: For tissues with extremely high biotin content, consider using non-biotin detection methods like direct fluorophore conjugation or other hapten-based systems (e.g., digoxigenin or DNP).

• Endogenous biotin controls: Include tissue sections processed without primary antibody but with the complete detection system to quantify background signal from endogenous biotin.

• Comparative validation: When studying samples from biotin deficiency models, validate results obtained with biotin-conjugated antibodies using unconjugated primary antibodies with secondary detection systems to confirm that observed changes in SLC19A3 levels (33% of baseline after 28 days of deficiency) are not artifacts of the detection method .

What protocols yield optimal results for Western blot detection of SLC19A3?

For optimal Western blot detection of SLC19A3, researchers should follow this validated protocol based on successful applications across multiple tissue types and cell lines:

Sample Preparation:

• Extract total protein from tissues or cells using standard lysis buffers containing protease inhibitors.

• Determine protein concentration using Bradford or BCA assay.

• Load 20-50 μg of total protein per lane for cell lines (SH-SY5Y, HepG2, HEK-293, HeLa) or tissue samples (liver, skeletal muscle, kidney, heart, placenta) .

Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels.

• Transfer to PVDF or nitrocellulose membranes using standard protocols.

Antibody Incubation and Detection:

• Block membranes in 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Incubate with primary SLC19A3 antibody at dilutions of 1:500-1:2000 in blocking buffer overnight at 4°C .

• Wash membranes thoroughly with TBST (3 × 10 minutes).

• Incubate with appropriate secondary antibody (e.g., goat polyclonal to rabbit IgG at 1:50000 dilution for rabbit primary antibodies) .

• Wash and develop using chemiluminescence detection.

Expected Results:

This protocol has been validated with multiple cell lines including SH-SY5Y, HepG2, HEK-293, and HeLa, ensuring reliable detection across various experimental models .

How should researchers validate SLC19A3 antibody specificity across different species?

Validating SLC19A3 antibody specificity across species requires systematic cross-reactivity testing due to potential sequence variations. Different commercial antibodies show variable cross-reactivity patterns, with some reacting with human, mouse, and rat samples , while others may be restricted to specific species .

Recommended Validation Protocol:

• Sequence alignment analysis: Compare SLC19A3 protein sequences across target species to identify regions of high conservation. For example, antibodies targeting the region within amino acids 150-300 or 191-282 of human SLC19A3 have demonstrated successful applications .

• Positive control testing: Validate antibody performance using known positive controls for each species:

  • Human: SH-SY5Y, HepG2, HEK-293, or HeLa cell lysates

  • Mouse: Liver, skeletal muscle, or kidney tissue lysates

  • Rat: Heart or liver tissue lysates

• Knockout/knockdown validation: Where available, use tissue or cells from SLC19A3 knockout models as negative controls. The Slc19a3-/- mouse model serves as an excellent negative control for antibody validation .

• Cross-reaction testing table: Document antibody performance across species and applications using a standardized table format:

This validation approach ensures reliable interpretation of results when conducting comparative studies across species, particularly important when investigating the differential sensitivity to inhibitors observed between human and murine THTR2 .

What controls should be included when using biotin-conjugated SLC19A3 antibodies in experimental designs?

When using biotin-conjugated SLC19A3 antibodies, implementing a comprehensive set of controls is essential to ensure experimental validity and accurate interpretation of results. The following controls should be included in your experimental design:

Technical Controls:

• Isotype control: Include a biotin-conjugated IgG of the same host species (e.g., rabbit IgG for rabbit polyclonal SLC19A3 antibodies) at the same concentration to assess non-specific binding .

• Endogenous biotin control: Process tissue sections with detection reagents only (streptavidin/avidin) without primary antibody to evaluate signal from endogenous biotin, particularly in biotin-rich tissues.

• Blocking control: Pre-incubate sections with excess unconjugated streptavidin to block endogenous biotin before application of biotin-conjugated antibodies.

Biological Controls:

• Positive tissue controls: Include known SLC19A3-expressing tissues or cells, such as HepG2 cells or liver tissue samples, in each experimental run .

• Negative tissue controls: Where available, include samples from SLC19A3 knockout models as definitive negative controls .

• Expression gradient controls: When studying biotin deficiency effects, include samples from various timepoints of biotin restriction to demonstrate the progressive decrease in SLC19A3 expression (e.g., samples at day 0 and day 28 of biotin restriction should show approximately 67% reduction in signal) .

Specificity Controls:

• Peptide competition: Pre-incubate the biotin-conjugated SLC19A3 antibody with excess immunogen peptide (e.g., recombinant human thiamine transporter 2 protein, amino acids 191-282) before application to verify binding specificity .

• Alternative antibody validation: Confirm key findings using a non-biotin-conjugated SLC19A3 antibody with a different detection system to ensure results are not artifacts of the biotin-avidin detection method.

Implementation of these controls ensures robust, reproducible results when using biotin-conjugated SLC19A3 antibodies across various experimental applications.